Abstract
β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.
Highlights
Mannan, one of the major hemicelluloses in higher plants, is primarily composed of b-1,4 linked D-mannose units
SACTE_2347 is a three-domain protein (Figure 2) that consists of a glycoside hydrolase family 5 (GH5) catalytic domain, a fibronectin type-III domain (Fn3, residues 362–437), and a carbohydrate binding module family 2 (CBM2) that are connected by two short linkers
GH5 Subfamily 8 The GH5 family is separated into 53 subfamilies, with each subfamily defined by sequence similarity, amino acid conservation, and experimentally determined biochemical properties [25]
Summary
One of the major hemicelluloses in higher plants, is primarily composed of b-1,4 linked D-mannose units. Galactoglucomannan, which is prevalent in pine wood, has glucose incorporated into the mannan chain and galactosyl branching. In addition to the incorporation of sugar, the C2 and C3 hydroxyl groups of both mannosyl and glucosyl unit of mannan are frequently acetylated [2]. Hydrogen bonding interactions between the galactosyl branches and the mannan chain along with the physical association of hemicellulose with cellulose makes deconstruction of hemicellulose and other plant cell wall polysaccharides a formidable task [3]. Enzymatic hydrolysis of mannancontaining polymers is essential for deconstruction of plant cell wall, softwoods such as pine
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